During the last decade colloidal gold particles have become popular as markers in cytochemistry applications. Colloidal gold particles may be prepared in a variety of sizes through the reduction of gold chloride, or similar compounds, with a variety of reagents, including white phosphorus, sodium ascorbate, or sodium citrate. Colloidal gold particles generally have an average diameter of from 1 to about 140 nm.
Colloidal gold particles are typically bound to proteins or immunoglobulins to form protein-colloidal gold complexes, these protein-colloidal gold complexes are useful for the visualization of immunoreactions. For example, protein-colloidal gold complexes are widely used as electron-dense markers for electron microscopy, as colored markers in light microscopy, as stains for identifying substances on blots, and as a visible indicator for the presence of various substances in solution, such as hormones in urine.
The adsorption of proteins onto the colloidal gold is variable, depending to a great extent on the reaction conditions. Reaction conditions which have been identified as being most important include the amount of colloidal gold, the concentration of protein, and the pH of the reaction solution.
It is desirable that protein-colloidal gold complexes have a uniform size, shape, charge, and ratio of charge to size. In other words, the complexes are preferably homogeneous. Homogeneity is an important factor in predicting the reactivity of complexes, and further the reproducibility of experimental results obtained using them. The term "homogeneous" or "homogeneity" as used herein is defined as relating to a product producing substantially symmetrical spots after subjection to electrophoretic migration on a flat bed gel electrophoresis system. Electrophoretically homogeneous protein-colloidal gold complexes, or a discrete fraction thereof, are defined in greater detail as occupying a discrete area in the gel, immediately following electrophoresis, which is (at least with one dimensional electrophoresis) no greater than the diameter of the round sample well plus about 10% (perpendicular to the electrophoretic migration path); such areas are preferably round, or one axis of the area may be slightly elongated but no more than about 1.34 times the diameter of the sample well (about 33% increase, parallel to the electrophoretic migration path). One aspect of homogeneity is related to the quantity of protein bound by the gold particles. It is known that the amount of protein attached to the colloidal gold likely influences any experimental results obtained. Therefore, a homogeneous preparation would be superior to one that is either unknown or known to be nonhomogeneous.
The testing and selection of homogeneous colloidal gold-protein complexes has in the past been largely restricted to only a few procedures including absorbance (1-3); passive gold agglutination (3); blotting (4-7); and centrifugation combined with direct observation in the electron microscope (8,9). The most commonly used methods for determining the homogeneity and usefulness of a particular preparation involves either a visual or spectrophotometric determination of the color change that occur when NaCI is added to the mixture. The spectrophotometer is used to determine the smallest absorbance or color change produced following the addition of NaCl to the protein-colloidal gold solution. These procedures are used not only to identify homogeneous preparation but also to select the pH and protein concentration that yield the lowest absorbance. Proponents of this method contend that solutions having a low absorbance contain minimal aggregates of complexes, and are therefore, homogeneous.
Visual and spectrophotometric methods essentially only distinguish between non-aggregated and aggregated preparations and do not identify the more subtle differences in the complexes which create heterogeneity. For example, more than one set of reaction conditions may be used to produce protein-colloidal gold complexes, which, according to spectrophotometric comparison, have identical or near identical absorbance, or degree of aggregation. However, it has been determined that the reactivity of these complexes is often different. Accordingly, visual or spectrophotometric methods cannot determine the homogeneity of a preparation. In addition, although direct observation with an electron microscope, centrifugation or chromatography can be used to identify, and in some cases even remove aggregates from a solution, unfortunately, however, these procedures do not detect more subtle differences between the particles themselves.
In light of the above deficiencies in the art, and the need for homogenous preparations of protein-colloidal gold complexes, it would be advantageous to provide a method for identifying the reaction conditions which produce homogeneous protein-colloidal gold complexes. Further, it would be advantageous to provide a method for producing homogeneous protein-colloidal gold complexes. Still further, it would be an advantage to provide a homogeneous protein-colloidal gold preparation. The present inventor has discovered that the electrophoretic mobility of protein-colloidal gold complexes can be used in the identification and production of homogeneous preparations. More particularly, it has been determined that complexes which exhibit an electrophoretic mobility which closely resembles that exhibited by the unbound free protein, are homogeneous.